1. Field of the Invention
The present invention relates to a novel antitumor protein hybrid, for example, cytotoxic against mouse tumors, and a process for the preparation thereof. More particularly, the present invention relates to a novel protein hybrid, which has a moiety which is substantially the fragment Fab of an anti-.alpha.-fetoprotein antibody and a moiety which is substantially the fragment A of a diphtheria toxin, is specifically useful as a remedy for malignant tumor e.g., in mice, and a process for the preparation of the same.
2. Description of the Prior Art
Many studies have been made on tumor-associated antigens and extensive research has especially been made with regard to the production of .alpha.-fetoprotein by liver cancer cells (Protein, Nucleic Acid, Enzyme, Vol.23, pp.579-593, 1978). In the body of an ordinary man, .alpha.-fetoprotein is, though formed during his fetal period, scarcely detected after his birth. However, when he suffers from liver cancer, its production takes place again in his body and its concentration in the blood reaches as high as 1,000 times the normal value. .alpha.-Fetoprotein is obtained from ascites fluid of a liver cancer patient and is purified by biochemical and immunochemical methods. Its properties are similar to those of serum albumin. It is a protein having a molecular weight of 70,000 and a isoelectric point of 4.5 to 5.0; however, its antigenicity is quite different from that of serum albumin. Therefore, it is possible to obtain an antibody (anti-.alpha.-fetoprotein antibody) specific Bind only to .alpha.-fetoprotein, by immunizing horses, cows, goats, sheep, monkeys or rabbits, etc. with purified .alpha.-fetoprotein. The antibody thus obtained binds to a cancer cell which produces .alpha.-fetoprotein; however, this specific binding can display only a slight cytotoxic activity against liver cancer cells. Accordingly, the present inventors discovered that a novel protein hybrid, which has a specific and strong cytotoxic activity against liver cancer cells, can be obtained by coupling an anti-.alpha.-fetoprotein antibody with fragment A of diphtheria toxin which has a strong cytotoxicity.
Several reports have been made on research for obtaining agents with selective cytotoxicity against cancer cells by conjugating an antitumor drug or a toxin to an antitumor antibody. Though no attempt has been made as to the use of an anti-.alpha.-fetoprotein antibody, some research has been made with the use of a diphtheria toxin as shown in the following examples.
For instance, F. L. Moolten et al. report that they prepared a conjugate by conjugating rabbit anti-SV40-transformed hamster sarcoma or lymphoma antibody to diphtheria toxin with glutaraldelyde as a coupling agent and were able to protect hamsters challenged with SV40-transformed tumors by administering the conjugate to hamsters (Journal of the National Cancer Institute, Vol.55, pp.473-477, 1975).
P. E. Thorpe et al. report that the conjugate prepared by coupling diphtheria toxin to antilymphocytic antibody by means of chlorambucil greatly reduced the protein synthesis of human lymphoblastoid cells, CLA4. (Nature, Vol.271, pp.752-754, 1978).
The results of these studies show that a conjugate of diphtheria toxin and antibody displays toxicity against the tumor cells selectively. However, these conjugates, when used as an antitumor drug, are believed to have some disadvantages as cited below. First, xenogeneic antibody (immunoglobulin) has a strong antigenicity in the human body and induces the formation of anti-xenogeneic immunoglobulin antibody which deactivates the antitumor activity and further causes an anaphylaxis shock. The second of the disadvantages is that the nonspecific toxicity of diphtheria toxin is not nullified. More particularly, the object of these methods is to concentrate diphtheria toxin on the surface of tumor cells by the aid of antitumor antibody; however, since the conjugate contains the whole molecule of diphtheria toxin in its composition, it is apt to bind to normal cell surface receptors for diphtheria toxin and display cytotoxicity against the normal cells. Thirdly comes the defect which is found in the method of cross-linking the antibody with the diphtheria toxin. Many of the cross-linking agents such as glutaraldehyde, toluene diisocyanate, chlorambucil, etc. effect the cross-linking not only between the antibody and the toxin but also between the antibody and the antibody, and the toxin and the toxin, and moreover, they effect the formation of intra-molecule bonds in the antibody and in the toxin molecule, thus causing the formation of undesirable products and decrease or loss of the antitumor activity.